The present invention relates a hexane bioactive fraction and obtained from the roots of an aromatic plant named Vetiveria zizanioides commonly found in India for inhibiting the growth of drug resistant bacterial infections in humans and animals. The invention also relates to a pharmaceutical composition comprising the bioactive extract with other additives for inhibiting the growth of drug resistant bacterial infections in humans and animals. The present invention also provides a process for the isolation of said bioactive extract.
Antibiotics have been used for long to cure bacterial, fungal and other infectious diseases of humans. Penicillin was the first antibiotic used against infections during the Second World War. Since then a number of antibiotics and their derivatives have been identified and used by man almost all of which were isolated from microbial sources. All the antibiotics in clinical use today can be grouped or classified according to their structure or functional groups. Streptomycin, kanamycin, tetracycline some of the well-known examples are aminoglycosides whereas penicillin and its derivatives are beta-lactam antibiotics. One of the commonly used antibacterials are quinolones or fluoroquinolones such as nalidixic acid, ciprofloxacin, norfloxacin etc. Fluoroquinolones are now widely used to treat urinary tract infections, upper respiratory tract infections, and tuberculosis, which are resistant to first-line drugs. However, many of the pathogenic bacteria such as Haemophilus influenzae, Neisseria Sp., Staphylococcus aureus, Escherichia coli are developing resistance to fluoroquinolone class of antibiotics limiting their clinical usefulness. Since, the mechanism of action of all the quinolones against bacteria is similar, development of resistance to one of the quinolone antibiotic would confer simultaneous cross-resistance to almost all the other quinolones also. Fluoroquinolones act by inhibiting the function of a bacterial enzyme DNA gyrase essential for the maintenance of supercoil nature of the bacterial chromosome. Resistance development is observed when a mutation in the DNA gyrase enzyme A subunit (GyrA+) specifically in the region called xe2x80x9cQuinolone Determining Region (QDR)xe2x80x9d occurs. The modified mutant form of A subunit (GyrAxe2x88x92) is incapable of binding to quinolone antibiotics and therefore is resistant. Such quinolone resistant infections are particularly difficult to cure. Kumar et al (Phytotherapy Research14: 14-15, 2000; U.S. Pat. No. 6,127,405) have identified a semi-synthetic plant compound xcex1-arteether which is capable of specifically killing quinolone drug resistant bacterial infections. The xcex1-arteether was obtained by etherification of artemissinin a sesquiterpene lactone compound from a Chinese medicinal plant Artemisia annua. In our effort to isolate and identify more potent plant compounds which are active against quinolone resistant bacteria we carried out a systematic bioactivity guided fractionation of the ethanolic extract prepared from the roots of Indian medicinal plant Vetiveria zizanioides. The subject mentioned below specifically describes the manner in which the compound inhibiting quinolone resistant bacteria was isolated and identified.
The main object of the invention is to develop a novel anti bacterial agent inhibiting the growth of multi drug resistant bacterial pathogens.
Another object of the invention is to provide a bioactive fraction from the roots of plant Vetiveria zizanioides. 
Another object of the invention is to provide a pharmaceutical composition comprising bioactive fraction or plant extract obtained from plant Vetiveria zizanioides 
Still another object of the invention is to provide a method of isolation of bioactive fraction from the roots of plant Vetiveria zizanioides. 
Accordingly, the present invention provides a bioactive hexane fraction named as CIM 109 obtained from the roots of plant Vetiveria zizanioides for inhibiting growth of multidrug resistant bacterial pathogens. The present invention also provides a pharmaceutical composition comprising bioactive fraction CIM 109 or plant extract or lyophilised extract to provide anti bacterial activity.
Accordingly, the present invention provides a bioactive hexane fraction CIM 109 obtained from the plant Vetiveria zizaniodes having inhibitory activity against multi drug resistant bacterial pathogens.
One embodiment of the invention, the said bioactive fraction inhibits the growth of bacterial pathogens which are resistant to nalidixic acid, oxolinic acid, sparfloxicin, ciprofloxicin, lomefloxicin and any other quinolones.
Another embodiment of the invention, the multidrug resistant bacteria is selected from the group consisting of genus Mycobacterium or Escherchia coli preferably selected from group consisting of Mucobacterium smegmatis MC2155, Pseudomonas aeruginosa, Bacillus subtilis MTCC-121, Mucobacterium smegmatis MC2155 Wld type, Mucobacterium smegmatis MC2155 (NaiR) 6b, Mucobacterium smegmatis MC2155 13a and E.Coli DH5a.
One more embodiment of the invention relates to a pharmaceutical composition for inhibiting the growth of the bacterial pathogens, comprising effective amount of bioactive fraction named CIM-109 or partially purified extract or lyophilised extract, obtained from the plant Vetiveria zizanioides. 
Another embodiment of the invention, the composition containing the said bioactive fraction is used singly or in combination thereof to the patient.
Still another embodiment, the composition may be administered systematically or orally and preferably orally.
Still another embodiment, the bioactive fraction is administered to the patient in combination with a pharmaceutically acceptable additives carriers, diluent, solvent, filter, lubricant, excipient, binder or stabiliser.
Yet another embodiment relates to the additive used which is selected from a group consisting of citric acid, calcium carbonate, magnesium hydroxide gel and/or gel and/or lactose.
Yet another embodiment relates to amount of active fraction in the composition is in the range of 100 mg to 500 mg.
Yet another embodiment of the invention relates to amount of composition administered to a subject is in the range of 500 mg to 1000 mg per day.
Yet another embodiment of the invention relates to amount of composition administered to a subject is preferably in the range of 150 mg to 700 mg per day.
Yet another embodiment of the invention, the subject is selected mammals, animals preferably humans.
In another embodiment of the invention provides a pharmaceutical composition useful for treating fluoroquinolone resistant bacterial infections including entenc and systemic infections, said composition comprising 10 to 50% by wt of root extract of vetiver, 0.4 to 1% by wt of citric acid, 10 to 20% by wt of calcium carbonate, 10 to 20% by wt of magnesium hydroxide gel, 20 to 60% by weight of lactose and optionally comprising other pharmaceutically acceptable additives. The above said composition can optionally compounded with honey by dispersing the constituents in honey.
One more embodiment of the invention relates to a method of treating patients with bacterial infection said method comprising administering a pharmaceutically effective dosage of bioactive fraction or a formulation comprising bioactive fraction or lyophilized extract of plant Vetiveria zizanioides thereof to the patient.
Another embodiment of the invention relates to a process for the isolation of bioactive fraction from the plant Vetiveria zizaniodes having inhibitory activity against multi drug resistant bacterial pathogens, the said process comprises steps of:
a) powdering the plant part of Vetivera Zizanioides, 
b) extracting the plant powder of step (a) by soaking in protic aqueous organic solvent for a period of 16-20 hours,
c) filtering the organic solvent extract of step (b),
d) evaporating the extract of step (c) under reduced pressure to remove the organic solvent to obtain an aqueous extract,
e) lyophilizing the aqueous extract of step (d) to get a powered extract,
f) dissolving the powdered extract of step (e) in 2% aqueous citric acid,
g) extracting the solution of step (f) successively with chloroform, n-butanol, methanol and finally with acetone to obtain respective organic extracts and an aqueous solution,
h) evaporating separately the organic extracts of step (g) to obtain respective residues,
i) neutralizing the aqueous solution of step (g) with ammonia solution,
j) testing the bioactivity of residues obtained in step (h) and neutralized solution of step (i) to identify residue from methanolic extract as bioactive residue,
k) macerating the residue of methanoloic extract of step (h) successively with hexane, chloroform and ethylacetate,
l) testing bioactivity of hexane, chloroform and ethylacetate fractions of step (k) to identify hexane fractions as a bioactive fraction,
m) purifying the residue of hexane fraction of step (l) on a silica gel column using eluant hexane and mixture of hexane-chloroform with increasing polarity, and
n) evaporating the hexane-chloroform (1:1) eluant fraction obtained from step (m) to yield a residue, which is purified by thin layer chromatography to achieve the required bioactive fraction.
Another embodiment of the invention, in which the bioactive fraction obtained in step (m), is designated as CIM 109.
Still another embodiment of the invention provides a bioactive fraction obtained from roots of Vetivera Zizanioides. 
Still another embodiment of the invention, the protic aqueous organic solvent used in step (b) theis selected from aqueous alcohol preferably aqueous ethanol.
In our study specifically directed at finding an antibiotic or plant compound, which can specifically kill quinolone drug resistant bacterial infections, we found that the ethanolic extract of roots of Vetiveria zizanioides was able to kill GyrAxe2x88x92 mutant E.coli bacteria but not the wild type strains (GyrA+). The ethanolic extract was then fractionated by liquid-liquid chromatography and the hexane fraction was to found to possess the growth inhibitory activity. The hexane fraction was then fractionated using silica gel column chromatography wherein the fraction eluted using 10% Chloroform in hexane indicated the desired bioactivity of eliminating Mycobacterium smegmatis (GyrAxe2x88x92) growth. Further to isolate and purify the active principle the eluted 10% chloroform in hexane fraction was separated by column chromatography. The Chl:Hex (50:50) fraction obtained from this column was able kill the GyrAxe2x88x92 strains of E.coli and M.smegmatis bacteria. The Chl:Hex (50:50) fraction was then purified by thin layer chromatography (TLC) to obtain a pure fraction called CIM 109 which has shown the desired bioactivty. Thus CIM 109 isolated from the roots of Vetiveria zizanioides was able to inhibit the growth of fluoro-quinolone resistant (GyrAxe2x88x92) bacteria. Hence, CIM 109 can be used for treating quinolone resistant bacterial infections of human and animals.